The objectives of the proposed research include a study of the mechanisms and regulation of neurotransmitter transport into synaptosomes and membrane vesicles derived from synaptosomes. Neurotransmitter agents in the mammalian central nervous system include gamma-aminobutyric acid (GABA), glutamate, aspartate, norepinephrine and choline (a precursor of acetylcholine). The use of membrane vesicles reconstituted with defined ion gradients permits a detailed examination of the Na ion-gradient hypothesis which states that the driving force for transport is the electrochemical sodium gradient. Experiments are planned to examine transport over a wide range of Na ion gradients (Na ion o is greater than Na ion i). In addition to uptake, the possible role of pre-synaptic muscarinic receptors on acetylcholine release will be tested. Experiments on the interaction of components of the cAMP-dependent protein kinase are planned. We propose to study the mechanisms of inactivation of the C subunit by chemical reagents which react with nucleophilic side chains of proteins. We also plan to study the interaction of C subunit and holoenzyme with nucleotides, protein, and polypeptide substrates. We specifically wish to test the notion that the holo-enzyme is inactive because it fails to interact with protein substrates. These ligand binding studies will be performed by fluorescence spectroscopy and by radio-ligand binding. We propose to study the mechanism of phosphorylation activation and inactivation of tyrosine hydroxylase. We plan to test the idea that tyrosine hydroxylase activity is affected directly by phosphorylation. An alternative notion, however, is that there are effectors present whose phosphorylation alters enzyme activity. The latter studies represent an extension of studies on the regulation of neurotransmitter biosynthesis.